The goal of the proposed project is to determine the underlined bioenergetic mechanisms of membrane related phenomena. The process of oxidative phosphorylation, electron transport and active transport have been treated separately; however, they all require intact membrane vesicles, oxidative energy and share certain common characteristics such as inhibition by certain uncoupling agents and specific respiratory inhibitors. However, the differences between the membrane-bound processes provide some insight concerning the mechanism(s) of each process and their possible interrelationship. Studies will be continued with membrane vesciles from Mycobacterium phlei which differ in vectorial orientation. Differences between oxidative phosphorylation and active transport have been observed following heat-treatment, D2O, freezing and thawing, sulfhydryl reagents, proton gradient and phospholipase treatment. The alterations of the membrane which effects ocidative phosphorylation or active transport is under investigation. Coupled phosphorylation and energized fluorescence were increased by heat-treatment of D2O whereas active transport was unaffected by such treatment. A study of the effect of phospholipase A, B, C and D may shed some light alterations in membrane structure which are caused by heat-treatment and effect the energized fluorescence. Removal of the membrane-bound coupling factor-latent ATPase (BCF4) results in a loss of phosphorylation, without affecting oxidation, and in a collapse of the proton gradient; however, the BCF4 depleted membranes are capable of active transport. These studies will be expanded to ghost preparations in which the membrane orientation is similar to that of the intact cell. Ghost preparations are cryptic for phosphorylation unless supplemented with a soluble factor. The nature and role of this soluble factor is under study. It would appear from these studies that the energy used for active transport is derived from the oxidation of substrates which establish a chemical gradient by the movement of Na ion ions. Studies of the uptake or efflux of Na ion ions will be undertaken. Malate-vitamin K reductase appears to be a model "artificial" membrane system. This system will be used to study conformational changes and the proton gradients (internal and external) which are generated during substrate oxidation.